This proposal concerns the regulation of ion-channel function by G-protein-coupled receptor (GPCR) signaling to membrane lipids. It focuses on the hypothesis that the function of many ion channels depends on the concentration of one rare phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane. The kinetics of signaling steps from M1 muscarinic receptors to phospholipase C will be measured to test the hypothesis that they are fast, perhaps reflecting a preformed signaling complex. The kinetics of the metabolic steps that deplete and replenish PIP2 will be measured to understand the cellular sources and dynamics of PIP2. All results will be fitted with a comprehensive kinetic model to provide additional information on the mechanisms of the signaling cascade. The ability of PIP2 concentration changes and muscarinic signaling to modulate function of KCNQ channels, several voltage-gated K+ channels (Kv channels), and voltage-gated Ca2+ channels (Cav channels) will be studied. The ability of arachidonic acid to modulate KCNQ and Cav channels will be analyzed. Physiological mechanisms for arachidonic acid production initiated by GPCR inputs will be defined. The methods will include patch-clamp electrophysiology, fluorescence resonance energy transfer, dynamic targeting of enzymes to cellular membranes, confocal microscopy, and chemical analysis. Most of the studies will be done on cell lines but a small number will be done in nerve cells of rodents to demonstrate the relevance to mammalian physiology. This work lays the basis for understanding hormonal control of mental state and the actions of many drugs of biological psychiatry. Many drugs of abuse and drugs of psychiatry act on the signaling systems studied here. The involuntary nervous system talks to its targets by the signaling mechanisms elucidated here.